The goal of the proposed studies is to address the role of one p53 family member, p63, in epithelial cell signaling and function, p63 is expressed in the basal layer of several stratified epithelial tissues including the epidermis, oral mucosa, esophagus, and bladder, as well as in several glandular structures such as bronchi, breast and prostate, p63-deficient mice and humans with p63 germline mutations display severe epithelium-related defects. The p63 gene encodes six splice variants with several predicted biochemical activities. In adults, expression of one p63 isoform, deltaNp63alpha is highest in the basal subpopulation of epithelial cells in tissues in which it is expressed and is frequently overexpressed in squamous cell carcinomas. Based on our recent discoveries that deltaNp63alpha: (i) can function as a transcriptional repressor, (ii) is regulated by growth stimulatory and inhibitory signaling, and (iii) loss can lead to epithelial cell senescence, we put forth the following interrelated hypotheses. DeltaNp63alpha is differentially phosphorylated by growth stimulatory and inhibitory signaling, and through transcriptional regulation of select target genes plays a role in maintenance of the proliferative state of epidermal reserve cell populations that separate the more differentiated cells from the stroma. We will test these hypotheses by analyzing deltaNp63alpha phosphorylation, identifying novel target genes that deltaNp63alpha binds and regulates in vivo, and determining the role of select p63 target genes in epithelial cell self-renewal. For our studies, we will predominantly use primary, normal, human epidermal keratinocytes and breast myoepithelial cells. New technologies and reagents will be exploited to test the hypotheses through the following Specific Aims: (i) To identify target genes that deltaNp63alpha binds and regulates in vivo; (ii) To determine the role of select deltaNp63alpha target genes in dictating cellular outcome under conditions of proliferation, senescence, differentiation, and stress; and (iii) To analyze deltaNp63alpha phosphorylation and the role it plays in regulating deltaNp63alpha protein levels and activity. The importance of understanding deltaNp63alpha regulation and function is underscored by the frequency of deltaNp63alpha overexpression in several human tumor types, the occurrence of human developmental disorders with germline mutations in p63, and the hope that modulating deltaNp63alpha signaling in human cancer cells may induce growth arrest or apoptosis.